Detection of Lethality Gene for Improved Fertility in Mammals

ABSTRACT

Oligonucleic acid molecules comprising a SNP site at a position corresponding to position 7480 of the bovine signal transducer and activator of transcription (STAT5A) coding sequence (SEQ ID NO: 1). Also disclosed are an array or a kit comprising the same, a method for detecting the SNPs, a method for progeny testing of mammals, a method for increasing human and non-human mammal pregnancy rate in natural and artificial reproduction processes. Further provided are cattle breeding methods for improved milk production traits.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for detectinga lethality allele in an animal, especially a mammal, and for improvingfertility or increasing reproductive performance of the animal. Thepresent invention further relates to methods and compositions forimproving milk production of dairy cattle.

BACKGROUND OF THE INVENTION

It is highly desirable in many contexts that reproductive performance inmammals be improved or enhanced. For example, in farm animals, increasedpregnancy rate and/or increased numbers of live offspring often wouldincrease profitability. In meat-producing animals, increased litter sizeand birth or hatching rates improve the overall efficiency andprofitability of a farm operation. Embryonic survival is directlyrelevant in avian species to improved hatching rates and for aquaticspecies to improved survival rate per spawn. Swine litter size would bepositively influenced by the elimination of conditions that are lethalfor developing embryos. Improved survival spreads the hatchery orpiggery cost over a larger number of offspring, for example day-oldchicks, post larvae shrimp, or piglets, and thus reduces the unit costof production.

In milk producing animals, aside from the inherent value of younganimals, periodic pregnancy and the resultant early lactation period arenecessary or desirable for the animal to have steady and high milkyield. Tremendous efforts, such as systematic animal breeding programsand artificial insemination, have been and continue to be invested inensuring that the animals have high and sustained productivity, and thatthe milk produced is of high quality or has desired composition.

While modern cattle breeding technologies have increased consistency ofherd quality or performance and generally achieved increased milk yield,many studies have reported a decrease in fertility in dairy cows. Cowswith the highest milk production have the lowest fertility performance.For example, it is well known that infertility is the major reason forculling cows, and it is estimated that in the UK alone, over 17,000 cowsare culled every year due to infertility or reproductive failure (GenusBreeding, UK). Epidemiological studies suggest that, in addition to milkproduction, other factors such as increasing levels of inbreeding areprobably decreasing reproductive efficiency in the dairy herd. Thefirst-service conception rate declined approximately from 65% in 1951 to40% in 1996 (Lucy, 2001, Reproductive loss in high-producing dairycattle: where will it end? J Dairy Sci. 84:1277-93.). A large number of“normal embryos” in dairy cattle are found to undergo early embryonicdeath, but there is currently no explanation for such early embryodeath. Reducing embryonic loss and achieving high rates of conception indairy cattle would change the way we manage the lactation cycle (Lucy,2001, supra).

The conception rate for cattle in the U.S. at first artificialinsemination (AI) has also been decreasing for many years, and accordingto one report it decreased by 0.45% per year over a 20-year period(Butler and Smith, 1989, J. Dairy Sci. 72:767-83.). There was anincrease in the number of AIs required for conception from 1.75 to morethan 3 over a period of 20 years (Lucy, 2001, supra). Conception ratesin large commercial herds stand at only 35-40% for mature cows. Asimilar need for improved reproductive performance exists with regard tomany other farm animals such as swine, equine, sheep and goat.

In humans, infertility or low fertility plagues a significant portion ofthe population. It has been reported that in Western countries, about10-15% of couples experience some difficulty with fertility (Evers,2002, Female sub-fertility. Lancet 2:151-159.) Many couples sufferingfrom impaired reproductive ability go to great effort and expense tosuccessfully give birth to a child. The economic and emotional costs ofembryonic mortality are significant, and a better understanding of itscauses and improved methods for managing it are needed.

Infertility or low reproductive performance in animals, however, ispresently poorly understood although it is known that there are manycontributing factors, both genetic and environmental. It is neverthelessreadily recognized that a genetic factor that causes the death of theembryo will be a major factor.

Lethal genes have been suggested as a cause of embryonic death and, ifpresent, could cause failures in recurrent inseminations. However,lethal genetic factors or lethality genes if dominant, cannot survive ina population. Consequently, little is known about these lethal geneticfactors. Identification and characterization of lethality genes wouldallow animal breeders, farmers and doctors to better understand lowfertility, selectively improve the chances of success in animalbreeding, develop strategic plans for improved fertility based on thegenetics of parents and help eliminate these lethal factors from thepopulation and improve overall reproductive performance in mammals.

As natural selection favors survival and reproduction of the moreadvantageous variants and elimination of the less advantageous variants,and an allele that confers lethality, even though recessive, generallydecreases reproductive fitness of the individual carrying the lethalityalleles. These recessive lethal alleles will eventually disappear fromthe population, unless it is otherwise selected for. Unnaturalprevalence of a lethal allele, that is, at a frequency higher thanpredicted, indicates that it is favored by the condition under which thepopulation is selected or propagated. Thus, if an allele is recessivelylethal (reproductively disadvantaged), yet confers certain desirableproduction traits (for example, in the case of dairy cattle, milk yieldor milk quality), this allele may be favored by breeding programs andpersist in incidences higher than expected under natural selectionconditions, even though its identity or phenotypic characteristics arenot known. Insight about the exact nature of the phenotypiccharacteristics of recessive lethal alleles will be invaluable inassisting animal breeders in balancing reproductive performance with theanimal's productive traits, and in achieving optimal economic outcome.

SUMMARY OF THE INVENTION

It has now been surprisingly discovered that a single nucleotidepolymorphism (SNP) in the STAT5 gene is responsible for early embryodeath in many animals including mammals, especially dairy cattle.Embryos homozygous with regard to a form of this SNP die at very earlystages. This is believed to be the first reported gene associated withlethality at an early developmental stage in mammals. It is furtherdiscovered that in dairy cattle, an individual heterozygous with regardto this allele produces higher milk yield, as well as higher milk fatcontent and protein content when compared to individuals homozygous ofthe non-lethal allele.

In one embodiment, the present invention provides an isolated single ordouble stranded nucleic acid molecule comprising a polymorphic site at aposition corresponding to position 7480 of exon 8 of the bovine SignalTransducer and Activator 5A (STAT5A) coding sequence (SEQ ID NO: 1),wherein position 7480 is either cytosine (the C allele) or guanine (theG allele), and at least about 9 contiguous nucleotides of SEQ ID NO: 1adjacent to the polymorphic site. Preferably, the nucleic acid moleculecomprises at least about 10, or at least about 15, or at least about 20contiguous nucleotides adjacent to the polymorphic site. The isolatednucleic acid molecule of the present invention in certain circumstancespreferably comprises not more than about 150 nucleotides, or not morethan about 100 nucleotides, or not more than about 50 nucleotides.

In a preferred embodiment, the polymorphic site is within 4 nucleotidesof the center of the nucleic acid molecule according to the presentinvention, or at the center of the nucleic acid molecule, or is at the3′-end of the nucleic acid molecule.

In another embodiment, the present invention provides an array ofnucleic acid molecules comprising the above isolated nucleic acidmolecule supported on a substrate. The array may further comprise one ormore markers in linkage disequilibrium with the polymorphic site. Inanother embodiment, the present invention provides a kit comprising anucleic acid molecule described above, and a suitable container.

In another embodiment, the present invention provides a method fordetecting single nucleotide polymorphism (SNP) on STAT5A coding regionin an animal cell, the method comprising determining the identity of anucleotide at a position corresponding to position 7480 of exon 8 ofbovine STAT5A coding sequence (SEQ ID NO: 1) of the cell. The animal maybe a mammalian, avian or aquatic species, and the animal cell may be anadult cell, an embryo cell, a sperm, an egg, a fertilized egg, or azygote. Preferably, the mammal is bovine.

In a preferred embodiment, the identity of the nucleotide is determinedby sequencing nucleic acid molecule, or a relevant fragment thereof,isolated from the cell. The nucleic acid molecule may be isolated fromthe cell via amplification by the polymerase chain reaction (PCR) ofgenomic DNA of the cell, or by RT-PCR of the mRNA of the cell.

In another preferred embodiment, the identity of the nucleotide isdetermined by hybridizing a suitable probe to a nucleic acid preparationfrom the cell, wherein the probe is preferably labeled with a detectablelabel.

In a further embodiment, the identity of the nucleotide is determined byan invasive signal amplification assay.

Preferably, the sequence of both copies of the polymorphic genetic locusin the cell is determined.

In another embodiment, the present invention provides a method whereinthe identity of the SNP site in the cell is determined based on thegenotypes of the parents, the genotypes of a daughter, or both.

In another embodiment, the present invention provides a method fordetermining whether an individual animal is suitable as a gamete donorfor natural mating, artificial insemination or in vitro fertilizationprocedure, the method comprising determining the allele identity of theSNP site according to the present invention, or of an allele in linkagedisequilibrium with the SNP site, and selecting as a gamete donor onlyan individual whose genotype is homozygous with regard to the C alleleat the SNP site, or homozygous with regard to an allele in linkagedisequilibrium with the C allele. Preferably, the animal is selectedfrom the group consisting of cattle, swine, equine, dog, sheep and goat.

In another embodiment, the present invention provides a method ofselecting an embryo for planting in a uterus, the method comprisingdetermining identify of the nucleotide at a position corresponding toposition 7480 of STAT5A (SEQ ID NO: 1) of the embryo while preservingthe viability of the embryo, and selecting for planting only an embryowhose genotype is CC homozygous at the position. Preferably, multipleovulation and embryo transfer (MOET) is used to generate multiplefertilized eggs.

In another embodiment, the present invention provides a method forincreasing successful pregnancy rate of a non-human animal, comprisingselecting a male or a female mammal for breeding purposes that are CChomozygous at a position correspond to position 7480 of exon 8 of bovineSTAT5A gene (SEQ ID NO: 1). Preferably, both the male and female parentsare selected to be CC homozygous. The female mammal may be in vitrofertilized.

In another embodiment, the present invention provides a method forincreasing pregnancy rate and reducing multiple pregnancy rate in ahuman assisted reproductive technologies (ART) procedure, the methodcomprising genotyping, via pre-implantation genetic diagnosis, thegenotype of embryos to be planted with regard to the nucleotidecorresponding to position 7480 of exon 8 of bovine STAT5A gene (SEQ IDNO: 1), and planting not more than 3 embryos which are homozygous CCwith regard to the position.

In yet another embodiment, the present invention provides a method fordetermining whether an individual dairy cattle is suitable as a gametedonor for a natural mating, artificial insemination or in vitrofertilization procedure, the method comprising determining alleleidentify of the SNP site according to the present invention, or of anallele in linkage disequilibrium with the SNP site, and selecting asgamete donor an individual whose genotype is heterozygous at the SNPsite, or heterozygous with regard to a locus in linkage disequilibriumwith the C allele. Preferably, an individual having CC genotype at theSNP site is selected to mate with an individual with a CG genotype.Still more preferably, gametes from the CG individual and the CCindividual are used in artificial insemination, or in in vitrofertilization, or in multiple ovulation and embryo transfer procedure.

In another embodiment, the present invention provides a method ofselecting a dairy cattle embryo for planting in a uterus, the methodcomprising determining the identity of the nucleotide at a positioncorresponding to position 7480 of bovine STAT5A (SEQ ID NO: 1) of theembryo while preserving the viability of the embryo, and selecting forplanting only an embryo whose genotype is CG heterozygous at theposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the partial sequence of the coding region for exons 5-19 ofSTAT5A (accession no. AJ237937) (SEQ ID NO: 3). The two PCR primers usedto amplify the fragment (bold) for initial SNP identification areunderlined, and the SNP position of 7480 is shaded. Positions are asoriginally labeled in the GenBank.

DETAILED DESCRIPTION OF THE INVENTION

The Signal Transducer and Activator (STAT) proteins are known to play animportant role in cytokine signaling pathways. The proteins aretranscription factors that are specifically activated to regulate genetranscription when cells encounter cytokines and growth factors, hencethey act as signal transducers in the cytoplasm and transcriptionactivators in the nucleus (Kisseleva et al., Signaling through theJAK/STAT pathway, recent advances and future challenges. Gene 285: 1-24(2002)). Binding of factors such as cytokines and growth factors tocell-surface receptors leads to receptor autophosphorylation at atyrosine, the phosphotyrosine being recognized by the STAT SH2 domain,which mediates the recruitment of STAT proteins from the cytosol andtheir association with the activated receptor. The STAT proteins arethen activated by phosphorylation via members of the JAK family ofprotein kinases, causing them to dimerize and translocate to thenucleus, where they bind to specific promoter sequences in target genes.In mammals, STATs comprise a family of seven structurally andfunctionally related proteins: Stat1, Stat2, Stat3, Stat4, Stat5a,Stat5b, and Stat6.

Signaling through the JAK/STAT pathway is initiated when a cytokinebinds to its corresponding receptor. This leads to conformationalchanges in the cytoplasmic portion of the receptor, initiatingactivation of receptor associated members of the JAK family of kinases.The JAKs, in turn, mediate phosphorylation at the specific receptortyrosine residues, which then serve as docking sites for STATs and othersignaling molecules. Once recruited to the receptor, STATs also becomephosphorylated by JAKs, on a single tyrosine residue. Activated STATsdissociate from the receptor, dimerize, translocate to the nucleus andbind to members of the GAS (gamma activated site) family of enhancers.

The seven STAT proteins identified in mammals range in size from 750 and850 amino acids. The chromosomal distribution of these STATs, as well asthe identification of STATs in more primitive eukaryotes, suggest thatthis family arose from a single primordial gene. STATs sharestructurally and functionally conserved domains (see e.g. Chen et al.,Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound toDNA. Cell 93: 827-839 (1998)).

The STAT5A protein is also known as the mammary gland factor (MGF). MGFknockout female mice failed to lactate. The encoding genomic region isabout 19,517 bp long, and has 19 exons. The bovine sequence is known andis publicly available in the GenBank (accession number AJ242522 forexons 1˜4 and AJ237937 for exons 5˜19. The protein was initiallyidentified in the mammary gland as a prolactin-induced transcriptionfactor. STAT5A is a member of the IFN-tau and placental lactogen (PL)signaling pathway. The uterus is exposed to IFN-tau, PL, as well asothers hormones including estrogen, progesterone, and placental growthhormone. Mediated by prolactin receptor (PRLR) homodimers, and perhapsby PRLR and growth hormone receptor (GHR) heterodimers, PL stimulatesthe formation of STATS homodimers, which in turn induce thetranscription of bovine uterine milk protein (UTMP) and osteopontin(OPN) genes (see e.g. Spencer T. E. and Bazer F. W. 2002. Biology ofprogesterone action during pregnancy recognition and maintenance ofpregnancy. Front. Biosci. 1, d1879-98; Stewart M. D., Choi Y., JohnsonG. A., Yu-Lee L.Y. et al. 2002. Roles of Stat1, Stat2, and interferonregulatory factor-9 (IRF-9) in interferon tau regulation of IRF-1. BiolReprod. 66, 393-400; Spencer T. E. and Bazer F. W. 2004. Conceptussignals for establishment and maintenance of pregnancy. Reprod BiolEndocrinol. 2, 49). The UTMP gene is known to affect milk productiontraits in cattle. The OPN protein was first described in 1979 as aprotein associated with malignant transformation, and has beenintensively studied in human, mouse, and sheep. It has been suggestedthat human OPN has various roles in cell adhesion, chemotaxis, cellsurvival, tissue remodeling, regulation of inflammation, fetal growthand development, and in initiating and maintaining pregnancy (Denhardtet al. 2001, Osteopontin as a means to cope with environmental insults:regulation of inflammation, tissue remodeling, and cell survival. J ClinInvest. 107:1055-1061; Johnson et al., 2003. Osteopontin: roles inimplantation and placentation. Biol Reprod. 69:1458-1471).

STAT5A and STAT5B from the same species share about 96% sequencesimilarity at the amino acid level. The sequence homology among STAT5proteins from different animal species is high, as shown in Table 1,which summarizes the sequence similarity of STATS proteins among variousspecies when compared to the bovine sequence. Table 1 also makes clearthat the sequence homology among mammalian species is especially high.TABLE 1 Sequence similarity among STAT5A proteins Sequence similarity toBovine Sequence Species Accession No. CAB52173 (%) Canine 97 Sus Scrofa96 Human 96 Mouse 96 Rattus 95 Ovies aries 94 Gallus gallus 90 Daniorerio 79 Takifugo rubripes 79 Xenopus laevis 87

The present inventor has identified a single nucleotide polymorphism(SNP) in STAT5A that is associated with early embryo death in animals.The term “polymorphism” as used herein refers to the occurrence of twoor more alternative genomic sequences or alleles between or amongdifferent genomes or individuals. “Polymorphic” refers to the conditionin which two or more variants of a specific genomic sequence can befound in a population. A “polymorphic site” is the locus at which thevariation occurs. A polymorphic site generally has at least two alleles,each occurring at a significant frequency in a selected population. Apolymorphic locus may be as small as one base pair, in which case it isreferred to as single nucleotide polymorphism (SNP). The firstidentified allelic form is arbitrarily designated as the reference form,and other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wild type form. Diploidorganisms may be homozygous or heterozygous for an allelic form. Abiallelic polymorphism has two forms, and a triallelic polymorphism hasthree forms, and so on.

Polymorphisms may provide functional differences in the geneticsequence, through changes in the encoded polypeptide, changes in mRNAstability, binding of transcriptional and translation factors to the DNAor RNA, and the like. Polymorphisms are also used to detect geneticlinkage to phenotypic variation.

SNPs have gained wide use for the detection of genetic linkage recently.SNPs are generally biallelic systems, that is, there are two allelesthat an individual may have for any particular SNP marker.

The SNP associated with early embryo death according to the presentinvention is located on STAT5A, at a position corresponding to position7480 on exon 8 of the bovine sequence (GenBank accession numberAJ237937) (see FIG. 1). It has been discovered that at this position,the predominant allele is cytosine (C), and the other allele is guanine(G), and that homozygote GG genotype is lethal and does not exist in thepopulation.

It is well-known to those ordinarily skilled in the art that the STAT5Agenes in all animal species are derived from a common ancestor, which isreflected in the high DNA sequence similarity of the gene among thesespecies. The nucleotide sequences of different animal species can beeasily aligned, using widely available sequence comparison/alignmenttools (e.g. Altschul, et al., 1990, “Basic local alignment search tool.”J. Mol. Biol. 215:403-410), allowing maximum number of identicalnucleotides on sequences from different animals to be positioned andcorrespond to each other. Similar alignment can be done based on theamino acid sequences encoded by the nucleic acid sequence. In manyinstances, appropriate gaps and insertions, determined by widelyaccepted computer algorithm, are introduced to allow for geneticdeletions or insertions that are believed to have occurred during theevolutionary history of the genes. Accordingly, by a “corresponding”nucleotide position, as used in the present invention, is meant anucleotide position that is identified using the nucleotide sequencealignment methodologies well-known in the art, based on a referencesequence, e.g. the bovine STAT5A sequence.

Using two PCR primers, STAT7 and STAT8, the inventor amplified andsequenced a 712-bp fragment from more than 2100 bovine samples fromdifferent cattle breeds (Holstein, Jersey, Brown Swiss, Bison bison, andBos indictis). STAT7 is located on exon 8, and STAT8 is located on exon9. The sequences of the two primers are: STAT7:5′-GAGAAGTTGGCGGAGATTATC-3′ (SEQ ID NO:1) and STAT8:5′-GTGTTCTCGTTCTTGAGCAG-3′ (SEQ ID NO: 2). It was found that at the SNPposition (7480), the predominant allele was cytosine (C), and the otherallele was guanine (G), with about 70% CC homozygote, and about 30% CGheterozygote. No cattle GG homozygote was found. This lead to theconclusion that the GG homozygote is lethal and does not exist in thepopulation.

The samples include semen samples from more than 1200 bulls and 1100blood samples from Holstein cows. The 1100 blood samples were obtainedfrom a University of Wisconsin (UW) daughter design resource population,consisting of 12 sire families. The sires used to create this populationwere chosen from a large number of candidate bulls with large numbers ofdaughters in production in the year 2000. Criteria for the finalselection of the 12 bulls included large numbers of daughters inproduction, in total and separately in lactations 1, 2 and 3, andrelatively low pedigree relationships among the chosen bulls in order tomore broadly sample the chromosomes of the U.S. Holstein population. The1200 semen samples were obtained from 30 half-sib families with agranddaughter design. Genomic DNA was extracted from semen samples bystandard methods using proteinase K and phenol/chloroform extraction,and from blood samples using GFX Genomic Blood DNA Purification Kit(Amersham Biosciences, Piscataway, N.J.). The DNA concentration wasmeasured using a spectrophotometer (Ultraspec 2100; AmershamBiosciences).

Cattle fetuses at 55-125 days were similarly genotyped and none wasfound to be GG homozygous, supporting the correlation between the GGhomozygote and lethality. Fetuses were obtained from a localslaughterhouse. DNA was extracted from fetal tissues and genotyped withSTATS gene.

Similar results were obtained from sheep samples. Two half-sib sheepfamilies were genotyped, where the two sires were heterozygous. Allgenotyped offspring (n=60) were either GC or CC.

Based on the results above, it was concluded that death of GG homozygousembryo occur at the first few days after fertilization.

The lethality of GG homozygote in cattle was further confirmed by anexperiment using IVF embryos. The inventor produced more than 300 JFVembryos using a GC sire and GC cows, or a GC sire and CC cows.Specifically, semen samples were obtained from seven bulls currentlyused in artificial insemination (Al) in the U.S., and their STAT5A genegenotyped. A bull that was found to be heterozygous (CG) was selectedfor the experiment. Oocytes were aspirated from 21 heterozygous (CG) andhomozygous (CC) cows and fertilized with semen obtained from theheterozygous bull. Survival rate of the embryos was measured at days7-9. It was found that embryos from the GC x CC parents had a survivalrate about 19% higher than that from the CGxCG parents. Genotyping thesurvived embryos revealed that all embryos were either CC or CG. Whendegenerative embryos (those that did not survive beyond day 5 or 6) weregenotyped, it was found that GG genotype was present, indicating thatthe GG genotype leads to early embryo degeneration.

The present inventors further tested the correlation between genotypesof the lethal gene and occurrence of pregnancy of Holstein heifers.Heifers is an ideal population to carry out this experiment compared tolactating cows because in lactating cows, genetic factors affectingpregnancy are believed to be diluted with many other environmentalfactors which makes it hard to detect these genetic factors. Incontrast, heifers have consistently higher pregnancy rates thanlactating cows and any small increase is of great economic importance.Records were collected of 623 inseminations between different bulls andcows. As shown in Table 2 below, the results were in agreement withprevious results obtained in the IVF, experiments. TABLE 2 PregnancyFrequency Heifers of Different Genotypes Frequency of pregnancy OPENPREGNANT CC genotype 0.34 0.66 GC genotype 0.45 0.55Pregnancy rate was 66% in cows carrying genotype CC versus 55% in cowscarrying genotype GC. This the first field experiment that confirms labresults.

The cattle and pig STAT5A proteins have 96% identity at the amino acidlevel, and the identity between sheep and pig STAT5 proteins is 92%.Because of this high similarity (see Table 1, supra), similar roles theprotein plays in various organisms (Development 130, 5257-5268 (2003))and its evolutionary history, a GG homozygote, or a similar SNP, inother animals, especially other mammals also have lethal effects on theembryos.

Based on the above results, the present invention provides, in oneembodiment, a method for increasing the reproductive performance of anmammal population, the method comprising determining the identity of thenucleotide, on both copies of the chromosome, at or corresponding toposition 7480 of exon 8 of the bovine STAT5A gene, and eliminatinganimals having a heterozygous CG genotype as a breeding parent. Asdiscussed above, while a GG homozygote cannot survive in the adultanimal population, a GC heterozygote will survive and be present in thepopulation. GC heterozygous animals, however, are not ideal candidatesas parents in a breeding program because about 25% of progenies fromsuch parents will be of the GG genotype and will not survive.

Accordingly, the present invention provides a nucleic acid based geneticmarker for identifying a lethality allele. This marker can be used forgenotyping an animal and for selecting an animal for breeding purposes.

In another embodiment, the present invention provides a method forselecting a mammal as a parent, wherein the mammal is genotyped asdescribed above, and selected as a breeder only if the animal ishomozygous CC.

In another embodiment, the present invention can be used to find markersthat are in strong linkage disequilibrium with the SNP corresponding toposition 7480 of the bovine STAT5A gene. These strongly linked markerscan be used as a substitute for the described marker. The presentinvention also provides, in a preferred embodiment, polymorphisms orpolymorphic sites that are in linkage disequilibrium with the SNPcorresponding to position 7480 of the bovine STAT5A gene. Linkage refersto the phenomenon that DNA sequences closely adjacent to each other inthe genome, specifically, on a chromosome in eukaryotes, have a tendencyto be inherited together. Typically, two polymorphic sequences areco-inherited because of the relative infrequency with which meioticrecombination events occur within the region between the twopolymorphisms, often due to physical proximity. Two sequences may alsobe linked because of some selective advantage of co-inheritance. Theco-inherited polymorphic alleles are said to be in linkagedisequilibrium with one another because, in a given population, theytend to either both occur together or else not occur at all in anyparticular member of the population. Where multiple polymorphisms in agiven chromosomal region are found to be in linkage disequilibrium withone another, they define a quasi-stable genetic “haplotype.” Incontrast, if meiotic recombination between two polymorphisms occursfrequently enough, the two polymorphisms will appear to segregateindependently and are said to be in linkage equilibrium.

Generally speaking, the frequency of meiotic recombination between twomarkers is proportional to the physical distance between them on thechromosome. However, there are “hot spots” and regions of repressedchromosomal recombination that cause discrepancies between the physicaldistance and the so-called “recombinational distance.” Consequently, agenetic haplotype could cover a broad region of the chromosome, withmultiple polymorphic loci in linkage disequilibrium with one another.Where one mutation or polymorphism is found within or in linkage withthis haplotype, another one or more polymorphic alleles of the haplotypecan be used as indicators of a phenotype known to be linked to or causedby the mutation. Such correlation can and often are used for prognosticor diagnostic procedures without the identification and isolation of theactual causal genetic factor. This is significant because the precisedetermination of the molecular nature involved in the genetic cause ofthe phenotype of interest can be difficult and laborious, and theavailability of polymorphic markers in linkage disequilibrium with thephenotype of interest often facilitates in the identification of thegenetic causal genetic factor(s).

In another embodiment, the present invention can be used to genotyperelatives of the animals of interest. Gene probability theory can thenbe used to predict the marker genotype of an individual based on markergenotype information from relatives in the population. For instance ifan individual animal has CG genotype and its female parent has CCgenotype, it will be known with 100% certainty that the male parent hasCG genotype without having genotyped that sire for the described marker.

In general, for use as markers, nucleic acid fragments, preferably DNAfragments, will be of at least 10 to 12 nucleotides (nt), preferably atleast 15 nt, usually at least 20 nt, often at least 50 nt. Such smallDNA fragments are useful as primers for the polymerase chain reaction(PCR), and/or probes for hybridization-based screening.

The present invention also encompasses the complementary sequencecorresponding to the polymorphism. In order to provide an unambiguousidentification of the specific site of a polymorphism, the numbering ofthe original sequences in the GenBank is shown in FIG. 1 and is used.

The term primer refers to a single-stranded oligonucleotide capable ofacting as a point of initiation of template-directed DNA synthesis underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as, DNAor RNA polymerase or reverse transcriptase) in an appropriate buffer andat a suitable temperature. The appropriate length of a primer depends onthe intended use of the primer but typically ranges from 15 to 30nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer needs not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with a template. Theterm primer site, or priming site, refers to the region of the targetDNA to which a primer hybridizes. The term primer pair means a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the DNA sequence to be amplified and a 3′, downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The term “probe” or “hybridization probe” denotes a defined nucleic acidsegment (or nucleotide analog segment) which can be used to identify byhybridization a specific polynucleotide sequence present in samples, thenucleic acid segment comprising a nucleotide sequence complementary ofthe specific polynucleotide sequence to be identified. “Probes” or“hybridization probes” are nucleic acids capable of binding in abase-specific manner to a complementary strand of nucleic acid.

An objective of genotyping according to the present invention is todetermine which embodiment of the polymorphism a specific sample of DNAhas. Many detection techniques are available and well-known to thoseskilled in the art. For example, an oligonucleotide probe can be usedfor such purpose. Preferably, the oligonucleotide probe will have adetectable label. Experimental conditions can be chosen such that if thesample DNA contains a C at position 7480, then the hybridization signalcan be detected because the probe hybridizes to the correspondingcomplementary DNA strand in the sample, while if the sample DNA containsan G, no hybridization signal is detected.

Similarly, PCR primers and conditions can be devised, whereby theoligonucleotide is used as one of the PCR primers, for analyzing nucleicacids for the presence of a specific sequence. These may be directamplification of the genomic DNA, or RT-PCR amplification of the mRNAtranscript of the genes. Amplification may be used to determine whethera polymorphism is present, by using a primer that is specific for thepolymorphism. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al (1990) Nucleic Acids Res. 18:2887-2890; andDelahunty et al (1996) Am. J. Hum. Genet. 58:1239-1246. The detectionmethod may also be based on direct DNA sequencing, or hybridization, ora combination thereof. Where large amounts of DNA are available, genomicDNA is used directly. Alternatively, the region of interest is clonedinto a suitable vector and grown in sufficient quantity for analysis.The nucleic acid may be amplified by PCR, to provide sufficient amountsfor analysis.

Hybridization may be performed in solution, or such hybridization may beperformed when either the oligonucleotide probe or the targetpolynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Oligonucleotides may be synthesized directly on the solid supportor attached to the solid support subsequent to synthesis. Solid-supportssuitable for use in detection methods of the invention includesubstrates made of silicon, glass, plastic, paper and the like, whichmay be formed, for example, into wells (as in 96-well plates), slides,sheets, membranes, fibers, chips, dishes, and beads. The solid supportmay be treated, coated or derivatized to facilitate the immobilizationof the allele-specific oligonucleotide or target nucleic acid. Forscreening purposes, hybridization probes of the polymorphic sequencesmay be used where both forms are present, either in separate reactions,spatially separated on a solid phase matrix, or labeled such that theycan be distinguished from each other. Assays may utilize nucleic acidsthat hybridize to one or more of the described polymorphisms, and mayinclude all or a subset of the polymorphisms listed in Table 1.

Hybridization may also be performed with nucleic acid arrays andsubarrays such as described in WO 95/11995. The arrays would contain abattery of allele-specific oligonucleotides representing a plurality ofthe polymorphic sites. One or both polymorphic forms may be present inthe array, for example the polymorphism at position 7480 of the STAT5Agene may be represented. Usually such an array will include at least 2different polymorphic sequences, i.e. polymorphisms located at uniquepositions within the locus. Arrays of interest may further comprisesequences, including polymorphisms, of other genetic sequences,particularly other sequences of interest. The oligonucleotide sequenceon the array will usually be at least about 12 nt in length, or mayextend into the flanking regions to generate fragments of 100 to 200 ntin length. For examples of arrays, see Ramsay (1998) Nat. Biotech.16:4044; Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart et al.(1996) Nature Biotechnol. 14:1675-1680; and De Risi et al. (1996) NatureGenetics 14:457-460. As well-known to those ordinarily skilled in theart, the presence or absence of hybridization signals, optionally incombination of the signal strength, will determine the presence orabsence of which of the alleles, and whether the sample is heterozygousor homozygous in regard to the SNP.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins whichrecognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variantalleles can be identified by single strand conformation polymorphism(SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries etal., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp.321-340, 1996) or denaturing gradient gel electrophoresis (DGGE)(Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al.,Proc. Natl. Acad. Sci. USA 86:232-236, 1989).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature and include the “Genetic BitAnalysis” method (WO 92/15712) and the ligase/polymerase mediatedgenetic bit analysis (U.S. Pat. No. 5,679,524). Related methods aredisclosed in WO 91/02087, WO 90/09455, WO 95/17676, U.S. Pat. Nos.5,302,509, and 5,945,283. Extended primers containing a polymorphism maybe detected by mass spectrometry as described in U.S. Pat. No.5,605,798. Another primer extension method is allele-specific PCR (Ruaoet al., Nucl. Acids Res. 17:8392, 1989; Ruao et al., Nudl. Acids Res.19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest.95:1635-1641, 1995). In addition, multiple polymorphic sites may beinvestigated by simultaneously amplifying multiple regions of thenucleic acid using sets of allele-specific primers as described inWallace et al. (WO 89/10414).

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

It is readily recognized by those ordinarily skilled in the art that inorder to maximize the signal to noise ratio, in probe hybridizationdetection procedure, the polymorphic site should be at the center of theprobe fragment used, whereby a mismatch has a maximum effect ondestabilizing the hybrid molecule; and in a PCR detection procedure, thepolymorphic site should be placed at the very 3′-end of the primer,whereby a mismatch has the maximum effect on preventing a chainelongation reaction by the DNA polymerase. The location of nucleotidesin a polynucleotide with respect to the center of the polynucleotide isdescribed herein in the following manner. When a polynucleotide has anodd number of nucleotides, the nucleotide at an equal distance from the3′ and 5′ ends of the polynucleotide is considered to be “at the center”of the polynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on. When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter,” and so on.

Alternatively, the relevant portion of the relevant genetic locus of thesample of interest may be amplified via PCR and directly sequenced. Itis readily recognized that numerous other primers can be devised basedon the sequence of Accession No. AJ 237937 such as those shown inFIG. 1. PCR and sequencing techniques are well known in the art andreagents and equipments are readily available commercially. The identityof the polymorphic site in the amplified fragment may also identified byRFLP, according to method and techniques well-known to those skilled inthe art.

Alternatively, an invasive signal amplification assay, as described ine.g. U.S. Pat. No. 5,422,253 and Lyamichev et al., 2000, Biochemistry39:9523-9532, both incorporated herein by reference in their entirety,may be used for detecting the SNP of interest. This assay takesadvantage of enzymes such as the 5′ nuclease activity of a DNApolymerase or the gene 6 product from bacteriophage T7 in their abilityto cleave polynucleotide molecules by recognizing specific structuresinstead of specific sequences. A single-stranded target molecule isannealed to a pilot oligonucleotide such that the 5′ end of the pilotforms a duplex with the target molecule. If the 3′ end of the pilotoligonucleotide does not pair with the target, a 3′ arm is formed. Whenexposed to a cleavage agent such as a DNA polymerase having a 5′nuclease activity or the gene 6 product from bacteriophage T7, thetarget molecule is cleaved in the 5′ region, one nucleotide into theduplex adjacent to the unpaired region of the target. If a cut in adouble-stranded molecule is required, the double-stranded molecule isdenatured. Because this unpaired 3′ arm can be as short as onenucleotide, this assay can be used for detecting a single-nucleotidedifference, e.g. in the context of SNP detection. The pilotoligonucleotide is designed such that it pairs perfectly with oneallele, but has a 3′, single nucleotide mismatch with another allele.Cleavage only occurs if there is a mismatch between the target moleculeand the pilot. To achieve signal amplification, the above invasivereaction is modified such that cleavage occurs on the pilotoligonucleotide. Two oligonucleotides are annealed in an adjacent mannerto the target molecule. The resulting adjacent duplexes overlaps by atleast one nucleotide to create an efficient substrate, called theoverlapping substrate, for the 5′ nucleases. The 5′ end of thedownstream oligonucleotide, also called the probe, contains an unpairedregion termed the 5′ arm (Lyamichev et al., 1993, Science 260:778-783.)or flap (Harrington and Lieber, 1994, EMBO J 13: 1235-1246) that is notrequired for the enzyme activity; however, very long arms can inhibitcleavage (Lyamichev et al., 1993, Science 260:778-783). Specificcleavage of the probe, termed invasive cleavage (Lyamichev et al., 1999,Nat. Biotechnol. 17 292-296; Kwiatkowski et al., 1999, Mol. Diagn. 4,353-364.), occurs at the position defined by the 3′ end of the upstreamoligonucleotide, which displaces or “invades” the probe. If the overlapbetween the adjacent oligonucleotides is only one nucleotide, cleavagetakes place between the first two base pairs of the probe, thusreleasing its 5′ arm and one nucleotide of the base paired region(Lyamichev et al., 1999, Proc. Natl. Acad. Sci. USA. 96: 6143-6148, andKaiser et al., 1999, J Biol. Chem. 274:21387-21394). If the upstreamoligonucleotide and the probe are present in large molar excess over thetarget nucleic acid, and invasive cleavage is carried out near themelting temperature of the probe, a cut probe can rapidly dissociate,and an intact probe will anneal to the target more frequently than willa cut probe, thus initiating a new cycle of cleavage. This allowsmultiple probes to be cut for each target molecule under isothermalconditions, resulting in linear signal amplification with respect totarget concentration and time (Lyamichev et al., 1999, Nat. Biotechnol.17: 292-296).

The present invention further provides a method for genotyping theSATA5A gene of an animal, especially a mammalian, or an individual of anavian or aquatic species, the method comprising determining thenucleotide identity for the two copies of the genetic locus. Oneembodiment of a genotyping method of the invention involves examiningboth copies of the genes or coding sequence of STAT5A, or a fragmentthereof, to identify the nucleotide pair at the polymorphic site in thetwo copies to assign a genotype to the individual. In some embodiments,“examining a gene” may include examining one or more of DNA containingthe gene, mRNA transcripts thereof, or cDNA copies thereof. As will bereadily understood by the skilled artisan, the two “copies” of a gene,mRNA or cDNA, or fragment thereof in an individual may be the sameallele or may be different alleles.

In another embodiment, a genotyping method of the invention comprisesdetermining the identity of the nucleotide pair at the polymorphic site.

The present invention further provides a kit for detecting the SNP ofthe present invention or for genotyping a sample, the kit comprising ina container a nucleic acid molecule, as described above, designed fordetecting the polymorphism, and optionally at least another componentfor carrying out such detection. Preferably, a kit comprises at leasttwo oligonucleotides packaged in the same or separate containers. Thekit may also contain other components such as instructional materialsand reagents (e.g. hybridization buffer where the oligonucleotides areto be used as a probe and/or enzymes for PCR or RFLP) packaged in aseparate container. Alternatively, where the oligonucleotides are to beused to amplify a target region, the kit may contain, preferablypackaged in separate containers, a polymerase and a reaction bufferoptimized for primer extension mediated by the polymerase, such as PCR.

In another embodiment the present invention provides an animal breedingmethod whereby genotyping as described above is conducted on animalembryos, and based on the results, certain embryos are either selectedor removed from the breeding program. In a preferred embodiment, whereCG parent(s) are not avoidable, the present invention provides aselective breeding method which takes advantage of multiple ovulationand embryo transfer procedure (MOET). The method comprisessuperovulating a female animal, collecting eggs from said superovulatedfemale, in vitro fertilizing said eggs using semen from a suitable maleanimal, implanting said fertilized eggs into suitable females allowingfor an embryo to develop, and determining the SNP of the developingembryo as described above. For animals other than dairy cattle, embryosother than homozygous CC are not transplanted or the pregnancyterminated depending on the circumstances. For dairy cattle, embryosother than CG genotype are not transplanted.

In another embodiment the present invention provides an animal breedingmethod whereby genotyping as described above is conducted on elite cows(cows to produce bulls), and based on the results, certain elite cowsare either selected or not selected to produce the next generation ofbreeding bulls (young bulls). This makes it possible to break anylinkage of the non-lethal allele with lower performance characteristicsthat may otherwise be present.

In another embodiment, the present invention provides a method forselecting a gamete donor in human assisted reproduction. Assistedreproductive technologies (ART) include IVF (in vitro Fertilization andembryo transfer), GIFT (gamete intrafallopian transfer) and ZIFT (zygoteintrafallopian transfer). A fertilized egg is transferred in ZIFT at thepronuclear stage, i.e., prior to the first cell division. The fertilizedegg undergoes the first cell division generally at about 30 hours afterfertilization, and becomes an embryo. The earliest stage embryo,generally up to four days after fertilization, and up to the 8-cellstage, is referred to as blastomere. From this point on, the embryo isreferred to as a morula, and is a solid mass of cells. Approximately 5-6days after fertilization, the embryo becomes a blastocyst which is ahollow ball of cells, filled with fluid. Embryo hatching andimplantation follow.

When a gamete (egg or sperm) donor is needed, the candidates aregenotyped and only those who are CC homozygous with regard to the STAT5ASNP of the present invention are selected, thereby eliminating thegeneration of GG embryos that will subsequently perish.

In the context of human ART, often one of the parents has a CGheterozygous genotype. The present invention provides a method andrelevant compositions that can be used to screen in vitro embryos forplanting in the uterus, increasing the rate of success while diminishingthe chance of multiple pregnancy. A naturally conceived embryo remainsin the fallopian tube for 4 days before entering the uterus andimplanting on or about day 6. Ideally embryos created through IVF shouldtherefore be transferred on Day 5. However for the last 10 years IVFembryos transferred on Day 5, after being cultured in conventionalgrowth media, had lower implantation rates than those transferred on Day3. For this reason the practice has been to transfer embryos into theuterus on Day 3. Implantation rates for Day 3 embryos, however, arestill only 15% to 20%. To achieve respectable pregnancy rates 3 or moreembryos must therefore be transferred whenever possible. Thisunfortunately dramatically increases the incidence of multiplepregnancies, which is the main criticism against IVF. Currently theability to accurately identify day 3 embryos that would survive andimplant from those that would not is lacking.

According to another embodiment of the present invention, a method isprovided for selecting early stage embryos for transfer, such thatembryos that cannot survive, i.e. GG homozygotes, are not selected ortransferred. This will ensure a high success rate, which would lead toavoidance of transfer of large numbers of embryos and multiplepregnancy, which is the main criticism of ART.

Pre-implantation genetic diagnosis (PGD) techniques are well known tothose ordinarily skilled in the art and are frequently used to testembryos for genetic disorders before it implants in the uterus.Typically, a single cell is removed from an 8-cell embryo through anopening in the outer protective coat. The procedure is carried out underthe microscope without damaging the embryo's ability to continue todevelop normally (because at this stage of development none of theembryo cells have become specialized). The cell is then analyzed for thepresence of genetic disorders.

The present invention further discovered, surprisingly, that dairycattle with heterozygous CG genotype has increased milk yield and highermilk fat and protein yield. Specifics of the experiments that lead tothis discovery are discussed below.

Populations and phenotypic data: Blood samples were obtained from theUniversity of Wisconsin (UW) daughter design resource population(henceforth: UW resource population). This population has beeninvestigated to search for genetic markers in association withsusceptibility to mycobacterium tuberculosis. The 12 sire families ofthis population were chosen from a large number of candidate bulls withlarge numbers of daughters in production in 2000. Blood samples from thebulls′ daughters have been collected through cooperation with commercialdairy producers throughout the U.S. since January 2001. Yield deviationdata for the UW resource populations for milk yield (kg), milk proteinand fat yields (kg), were obtained from the USDA Animal ImprovementPrograms Laboratory (Beltsville, Md.). Summary statistics of these datafor milk production traits are given in Table 3. TABLE 3 Means, standarddeviations (SD), minimum, and maximum yield deviations (YD) of cows inthe UW resource population for the production traits University ofWisconsin resource population Trait Mean (Kg) SD Min Max Milk YD 1092.61815.1 −5917 7344 Fat YD 29.97 68.17 −277 322 Protein YD 29.86 48.73−159 181

Polymorphism detection and genotyping: Genomic DNA was extracted fromblood samples using GFX Genomic Blood DNA Purification Kit (AmershamBiosciences, Piscataway, N.J.). DNA concentration was measured using aspectrophotometer (Ultraspec 2100; Amersham Biosciences). Amplificationwas performed in a 25 μl reaction volume, which included 50 ng genomicDNA, 50 ng each primer, 200 μM each dNTP, 2.5 μl 10× PCR buffer(Promega, Madison, Wis.), and 0.3 u Taq DNA polymerase (Promega). Thetemperature cycles were as follows: 95° C. for 5 min, followed by 30cycles of 94° C. for 45 s, touchdown annealing from 65-50° C. for 45 s(<2° C./cycle), 72° C. for 45 s, and a final extension at 72° C. for 7min. For individual genotyping, primers STAT7 and STAT8 were used toamplify 50 ng genomic DNA and the PCR products were digested with therestriction enzyme BstII that distinguishes alleles C and G of the SNP.The digestion products were electrophoresed on a 1.5% agarose gel.

Statistical Analysis: Sires and their daughters are genotyped, andphenotypic data was available for the daughters. The linear model usedwasY _(ijk)=μ+genotype_(i)+sire_(j)+Map+e_(ijk)where Y_(ijk) is the yield deviation (milk, fat, protein) of daughter k,μ is the mean, genotypei is the effect of genotype i, sire_(j) is thesire j effect, Map is M paratuberculosis infection status(noninfected=0, infected=1), and e_(ijk) is the residual.

The results are shown in Table 4 below: TABLE 4 Estimates of the effectsof CG genotypes, standard errors (SE) and P-values for milk productiontraits (kg) as a deviation from the effect of the genotype (CC) in theUW resource population Milk yield ± Fat yield ± Protein yield ± GenotypeSE (P) SE (P) SE (P) CG 445.9 ± 179.4 14.53 ± 7.00 9.65 ± 4.86 (0.0133)(0.0386) (0.073)

Table 4 shows the estimates of the genotype effects for milk yield,protein and fat yield in the UW resource population. Compared togenotype CC, genotype CG was associated with a significant increase inmilk yield (445.9 Kg), fat yield (14.53 Kg) and protein yield (9.65 Kg).

Accordingly, in another embodiment, the present invention provides amethod of improving dairy cattle breeding. Specifically, because anideal genotype for a milk-producing cow is CG, for both survivabilityand production traits, a natural mating between two CC parents is notdesirable. Because a GG parent does not exist, CC×GG (which wouldproduce 100% CG progeny) is not possible. Thus, parents should beselected for CG×CG matings or CC×CG matings. Both give 50% CG progeny,and 25% CC. CC×CG matings are particularly preferred because it is moredesirable for the remaining 25% of the resultant embryos to have a CCgenotype, than 25% GG for them to have genotype which will die at veryearly stages of the embryo development, which will occur in a CG×CGmating. In addition, CC×CG mating is preferred because milk productiontraits may be otherwise compensated by additional genetic traits.

In situations where there are more control, for example in in vitrofertilization and embryo transfer, the present invention furtherprovides a method of selecting parents (CG), such that fertilizationoccurs between CC×CG or CG×CG parents, in combination with a step tode-select CC embryos (poor production traits) and GG embryos (earlyembryo death), so that only CG embryos are planted.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof. Allreferences cited hereinabove and/or listed below are hereby expresslyincorporated by reference.

1. An isolated single or double stranded oligonucleotide moleculecomprising a polymorphic site at a position corresponding to position7480 of exon 8 of bovine Signal Transducer and Activator 5A (STAT5A)coding sequence (SEQ ID NO: 1), wherein position 7480 is cytosine orguanine, and at least about 9 contiguous nucleotides of SEQ ID NO: 1adjacent to the polymorphic site.
 2. The oligonucleotide moleculeaccording to claim 1, which comprises at least about 15 contiguousnucleotides adjacent to the polymorphic site.
 3. The oligonucleotidemolecule according to claim 2, which comprises at least about 20contiguous nucleotides adjacent to the polymorphic site.
 4. Theioligonucleotide molecule according to claim 1, which comprises not morethan about 150 nucleotides.
 5. The oligonucleotide molecule according toclaim 1, which comprises not more than about 100 nucleotides.
 6. Theoligonucleotide molecule according to claim 1, which comprises not morethan about 50 nucleotides.
 7. The oligonucleotide molecule according toclaim 1, wherein the polymorphic site is within 4 nucleotides of thecenter of the oligonucleotide molecule.
 8. The oligonucleotide moleculeaccording to claim 7, wherein the polymorphic site is at the center ofthe oligonucleotide molecule.
 9. The oligonucleotide molecule accordingto claim 1, wherein the polymorphic site is at the 3′-end of theoligonucleotide molecule.
 10. An array of nucleic acid moleculescomprising the isolated oligonucleotide molecules according to claim 1supported on a substrate.
 11. The array according to claim 10, furthercomprising one or more markers in linkage disequilibrium with thepolymorphic site.
 12. A kit comprising a nucleic acid molecule of claim1, and a suitable container.
 13. A method for detecting singlenucleotide polymorphism (SNP) on STAT5A coding region in an animal cell,the method comprising determining the identity of a nucleotide at aposition corresponding to position 7480 of exon 8 of bovine STAT5Acoding sequence (SEQ ID NO: 1) of the cell.
 14. The method according toclaim 13, wherein the animal is a mammalian, avian or aquatic species.15. The method according to claim 13, wherein the animal cell is anadult cell, an embryo cell, a sperm, an egg, a fertilized egg, or azygote.
 16. A method according to claim 14, wherein the animal is amammal.
 17. A method according to claim 13, wherein the mammal isbovine.
 18. A method according to claim 13, wherein the identity of thenucleotide is determined by sequencing a nucleic acid moleculecomprising a position corresponding to position 7480 of exon 8 of bovineSTAT5A coding sequence, or a relevant fragment thereof, isolated fromthe cell.
 19. A method according to claim 18, wherein the nucleic acidmolecule is isolated from the cell via amplification by the polymerasechain reaction (PCR) of genomic DNA of the cell, or by RT-PCR of themRNA of the cell.
 20. A method according to claim 13, wherein theidentity of the nucleotide is determined by hybridizing a suitable probeto a preparation comprising the nucleic acid from the cell.
 21. A methodaccording to claim 20, wherein the probe is labeled with a detectablelabel.
 22. A method according to claim 13, wherein the identity of thenucleotide is determined by an invasive signal amplification assay. 23.A method according to claim 13, wherein the identity of the nucleotideof both copies of the coding sequence of the cell is determined.
 24. Amethod according to claim 12, wherein the identity of the nucleotide ofboth copies of the sequence is determined based on genotypes of theparent of the cell, genotypes of the daughter of the cell, or both. 25.A method for determining whether an individual animal is suitable as agamete donor for natural mating, artificial insemination or in vitrofertilization, the method comprising determining allele identify of theSNP site according to claim 13, or of an allele in linkagedisequilibrium with the SNP site, and selecting as gamete donor anindividual whose genotype is homozygous with regard to C allele at theSNP site, or homozygous with regard to an allele in linkagedisequilibrium with the C allele.
 26. The method according to claim 25,wherein the animal is selected from the group consisting of cattle,swine, equine, dog, sheep and goat.
 27. The method according to claim25, wherein the animal is human.
 28. A method of selecting an embryo forplanting in a uterus, the method comprising determining identify of thenucleotide at a position corresponding to position 7480 of bovine STAT5A(SEQ ID NO: 1) of the embryo while preserving the viability of theembryo, and selecting for planting only an embryo whose genotype is CChomozygous at the position.
 29. The method according to claim 28,wherein the animal is an animal selected from the group consisting ofcattle, swine, equine, dog, sheep and goat.
 30. The method according toclaim 29, wherein multiple ovulation and embryo transfer (MOET) is usedto generate multiple fertilized eggs.
 31. The method according to claim28, wherein the animal is human.
 32. A method for increasing successfulpregnancy rate of a non-human animal, comprising selecting a male or afemale mammal for breeding purposes that are CC homozygous at a positioncorrespond to position 7480 of exon 8 of bovine STAT5A gene (SEQ ID NO:1).
 33. A method according to claim 32, wherein both male and femaleparents are CC homozygous.
 34. The method according to claim 33, whereinthe female mammal is in vitro fertilized.
 35. A method for increasingpregnancy rate and reducing multiple pregnancy rate in a human ARTprocedure, the method comprising genotyping, via pre-implantationgenetic diagnosis, the genotype of embryos to be planted with regard tothe nucleotide corresponding to position 7480 of exon 8 of bovine STAT5Agene (SEQ ID NO: 1), and planting not more than 3 embryos which arehomozygous CC with regard to the position.
 36. A method for determiningwhether an individual dairy cattle is suitable as a gamete donor fornatural mating, artificial insemination or in vitro fertilization, themethod comprising determining allele identify of the SNP site accordingto claim 13, or of an allele in linkage disequilibrium with the SNPsite, and selecting as gamete donor an individual whose genotype isheterozygous at the SNP site, or heterozygous with regard to a locus inlinkage disequilibrium with the SNP site.
 37. The method according toclaim 36, wherein an individual having CC genotype at the SNP site isselected to mate with an individual with a CG genotype.
 38. The methodaccording to claim 37, wherein gametes from the CG individual and the CCindividual are used in artificial insemination, or in in vitrofertilization, or in multiple ovulation and embryo transfer procedure.39. A method of selecting a dairy cattle embryo for planting in auterus, the method comprising determining identify of the nucleotide ata position corresponding to position 7480 of bovine STAT5A (SEQ IDNO: 1) of the embryo while preserving the viability of the embryo, andselecting for planting only an embryo whose genotype is CG heterozygousat the position.